Direct radiolabeling of antibodies and other proteins with technetium or rhenium

ABSTRACT

Proteins containing one or more disulfide bonds are radiolabeled with radionuclides of technetium or rhenium for use in diagnosis and treatment of a variety of pathologic conditions. Radiolabeling is accomplished by partial reduction of the disulfide bonds of the protein using Sn (II), or using other reducing agents followed by the addition of Sn (II), removal of excess reducing agent and reduction by-products, and addition of a specified amount of pertechnetate or perrhenate reducing agent, such as stannous tartrate, with the addition accomplished in such manner that further reduction of the protein is limited. The resulting product may be stored frozen or lyophilized, with radiolabeling accomplished by the addition of pertechnetate or perrhenate solution.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part application of U.S. patentapplication Ser. No. 07/391,474, filed Aug. 9, 1989, entitledRadiolabeling Antibodies and Other Proteins with Technetium or Rheniumby Regulated Reduction, .Iadd.now U.S. Pat. No. 5,032,237 .Iaddend.theteachings of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

This invention relates to a method, composition and kit forradiolabeling proteins, including antibodies, with radioisotopes oftechnetium and rhenium such as tech- netium-99m.

2. Description of the Related Art Including Information Disclosed under37 C.F.R. Sections 1.97-1.99 (Background Art)

The use of radioisotopes to label proteins is well known. Thesecompositions can be used in assays, can be administered to the humanbody to visualize or monitor functioning of various parts of the body orto determine the presence and location of particular antigens,antibodies, hormones and the like, and can be used in the treatment ofvarious disease states. A variety of radioisotopes, including isotopesof iodine, technetium, indium, and rhenium have been used. It is alsowell known that protein molecules can be tagged or labeled withtechnetium-99m to form a diagnostic or imaging agent.

Technetium-99m has been utilized to radiolabel proteins, chelatingagents, phosphonate bone scanning compositions and the like by atechnique which utilizes sodium pertechnetate wherein the technetiuminitially is in the +7 state. Technetium-99m is generally available onlyas sodium pertechnetate. The pertechnetate must be contacted with areducmg agent, such as stannous chloride, in order to reduce thetechnetium to the +3, +4 or +5 oxidation state in the presence of theprotein, chelating agent or like substance which is to be radiolabeled.The technetium must be maintained in this reduced state in order tomaintain the chemical bond between the technetium molecule and thesubstrate being radiolabeled. It is also necessary that the technetiumbe firmly bound to the protein such that the reduced technetium is nottransferred to other molecules or other proteins present in the assay,patient's blood or other media in which the radiolabeled substance willbe utilized.

Several different methods have been utilized to radio-label proteins,particularly monoclonal antibodies, with technetium-99m. The methodsinvolve two general approaches. One approach is indirect in which abifunctional chelating agent is attached to the protein via onefunctional group and the technetium-99m is attached via the otherfunctional, or chelating group. This method was introduced by Krejcarek.G. E. and Tucker. K. L. ("Covalent Attachment of Chelating Groups toMacromolecules," Biochemical and Biophysical Research CommunicationsVol. 77, pp. 581-585, 1977) and has been widely employed in manyvariations using a wide variety of bifunctional chelating agents such asdescribed in the review of Wensel and Meares (Wensel, T. G., and Meares,C.F., "`Bifunctional` Chelating Agents for Binding Metal Ions toProteins," Radioimmunoimaging and Radioimmunotherapy, S. W. Burchiel andB. A. Rhodes, eds., Elsevier Publishing Co., New York, pp. 185-196,1983). Other methods are disclosed by Hnatowich, D. J., U.S. Pat. Nos.4,668,503 and 4,479,930, by Haber, E., and Khaw, B. A., U.S. Pat. No.4,421,735, by Fritzberg, A. R., and Kasina, S., U.S. Pat. No. 4,670,545and by Baidoo, K. E., et al, "^(99m) Tc Labeling of Proteins: IrtitialEvaluation of Novel Diaminedithiol Bifunctional Chelating Agent," CancerRes (Supp), Vol. 50, pp. 799s-803s, 1990. The bifunctional chelatemethods all present significant limitations, including the complexity ofthe radiolabeling procedure, the time required to accomplishradiolabeling, and the introduction and presence of substances which mayaffect the protein.

The other general approach is direct labeling. Although several directmethods have been reported, the fast direct method capable of providinga sufficiently strong bond between the protein and the technetium-99mfor in vivo applications was the direct or pretinning method describedin U.S. Pat. No. 4,424,200, entitled Method for Radiolabeling Proteinswith Technetium-99m, to Crockford, D. R., and Rhodes, B. A. In thismethod, a single reduction solution, consisting of stannous [Sn (II)]chloride with tartrate and phthalate salts, is used. This solutionserves to (1) reduce the protein, thereby exposing reactive sulfidegroups, (2) protect the reactive sulfide groups of the reduced proteinto prevent reformation of disulfide bonds, (3) reduce sodiumpertechnetate, and (4) complex the reduced Tc-99m and transfer it to thesulfide binding sites of the reduced protein. With this method, manyproteins can be successfully radiolabeled with technetium-99m. Severalinvestigators have reported on the use of this method (Rhodes, B. A., etal, "Technetium-99m Labeling of Murine Monoclonal Antibody Fragments,"Journal of Nuclear Medicine, Vol. 27, pp. 685-693, 1986: Some, P., etal, "Radioimmunoimaging of Experimental Thrombi in Dogs UsingTechnetium-99m-Labeled Monoclonal Antibody Fragments Reactive with HumanPlatelets," Journal of Nuclear Medicine, Vol. 27, No. 8, pp. 1315-1320,1986).

Other early direct labeling methods were reported, but did not yield astable Tc-protein bond. The reason for the instability of the Tc-proteinbond in prior methods (Stern, H. S., et al., RadioactivePharmaceuticals, U.S. Atomic Energy Commission (1966), Textbook, Chapter19, pp. 359-375; Lin, M. S., et al., "Use of Fe (II) or Sn (II) Alonefor Technetium Labeling of Albumin," Journal of Nuclear Medicine, Vol.12, No. 5, pps. 204-211, 1970; Eckelman, W. C., et al., ^(99m) Tc-HumanSerum Albumin," Journal of Nuclear Medicine, Vol. 12, No. 11, pp.707-710, 1971; Wong, D. W., et al., "A Rapid Chemical Method of LabelingHuman Plasma Proteins with ^(99m) Tc-Pertechnetate at pH 7.4,"International Journal of Applied Radiation and Isotopes, Vol. 29, pp.251-253, 1978; Colombetti, L. G., et al., "A Rapid Method for LabelingIgG with 99m-Tc," Journal of Nuclear Medicine, Vol. 20, p. 652, 1979;Rhodes, B. A., U.S. Pat. No. 4,305,922, Labeling Proteins with 99m-Tc byLigand Exchange, was that the protein had not been reduced to providereactive sulfide groups which are necessary for the formation of strongbonds between the protein and the reduced radionuclide.

Subsequently, a number of methods have been reported which employvariations on the method of U.S. Pat. No. 4,424,200. These variationsgenerally involve one or more of the following: (1) Disulfide reducingagents other than Sn (II), such as 2-mercaptoethanol, dithiothreitol andtheir analogs, are used to reduce the protein; (2) reactive sulfidegroup protecting agents other than Sn (II), such as Zn (II), areemployed; (3) pertechnetate reducing agents other than Sn (II), such asdithiothreitol, are used; and (4) complexing agents other than tartrate,such as phosphonates, are used to bind the reduced technetium andtransfer it to the sulfide groups of the protein.

These methods have generally not resulted in any improvement over themethod of U.S. Pat. No. 4,424,200. None of the methods disclosed yieldresults comparable to those achieved with the method, composition andkit disclosed herein.

Schwarz, A., and Steinstruaber, A., "A Novel Approach to Tc-99m-LabeledMonoclonal Antibodies," Journal of Nuclear Medicine, Vol. 28, p. 721,1987, and Bremer, K. H., et al., European Patent Office Application No.0 271 806 A2 filed Dec. 8, 1987), reduce the disulfide groups of theprotein with monothiols, such as 2-mercaptoethanol or2-mercaptoethylamine. Sn (II) is used to reduce the pertechnetate andthe reduced technetium is complexed with phosphonates or pyrophosphates.This method requires two or more vials and multiple steps to achieveradiolabeling. The phosphonates used can give rise to radiocolloidimpurities in the final product. In addition, the chemicals used toreduce the disulfide groups, such as 2-mercaptoethanol, are potentiallytoxic.

Reno, J. W., et al., U.S. Pat. No. 4,877,686 Radionuclide AntibodyCoupling and European Patent Office Application No. 0 237 150 (filedJan. 19, 1987), used dithiothreitol (DTT) to reduce the disulfide groupsof the protein, then protect the reactive sulfides with Zn (II) or othersulfhydryl group derivatizing reagents. They use tartrate salts tocomplex and transfer the reduced radionuclide. This method usespotentially toxic chemicals, such as dithiothreitol, to reduce theantibody. It also requires multiple steps to radiolabel the protein.

Pak, K. Y., et al, "A Rapid and Efficient Method for Labeling IgGAntibodies with Tc-99m and Comparison to Tc-99m Fab' AntibodyFragments," Journal of Nuclear Medicine, vol. 30, p. 793, 1989, andPatent Cooperation Treaty International Patent Application No. WO88/07382 (filed Apr. 1, 1988), used dithiothreitol to reduce theantibodies. Tartrate or glucoheptonate salts and their analogs are addedto complex and transfer the reduced radionuclides. This method also usespotentially toxic chemicals, and requires multiple steps to radiolabel.

Shochat, D., et al., European Patent Office Application No. 0 336 678(filed Apr. 3, 1989) use conventional disulfide reducing agents, such ascysteine, dithiothreitol, 2-mercaptoethanol, dithionite or the like.They claim that the pretinning method of U.S. Pat. No. 4,424,200 doesnot work well, indicating that some of the radiometal is bound to siteswhich are comparatively labile in the presence of blood or other bodilyfluids or tissues. They give a single example in the application,preparation of Tc-99m-anti-CEA-Fab', which example appears to be exactlythat of the '200 patent. The use of Sn (II) to reduce sodiumpertechnetate is well known in the prior art, and is disclosed in the'200 patent and other references.

The advantage of the Sn (II) method of reducing disulfides of proteinsover other methods is that the Sn (II) both reduces the bond andcomplexes with the sulfide formed by the reduction to protect thesulfide from reverting to unreactive disulfide. When organic reducingagents such as DTT are used to reduce the disulfide groups of theprotein, the reducing agent must be removed before sulfide protectinggroups are added, otherwise the protecting groups will react, usually byformation of a precipitate, with the reducing agent. If the reducingagent is first removed to avoid this reaction between it and the sulfideprotecting agent, then the reduced protein is left for a period of timein which the reactive sulfide groups can reform unreactive disulfidebonds. The Sn (II) reduction method, described in this invention, is newin that it permits simultaneous reduction and complexing of disulfides.

Other methods of direct labeling have also been reported which differchemically from the four-step process described above. Paik, C. H. etal., U.S. Pat. No. 4,652,440, Method of Stably Radiolabeling Antibodieswith Technetium and Rhenium, label proteins by Sn (II) reduction in thepresence of a strong chelating agent, DTPA, which competes for thereduced radionuclide. Only strongly bonded Tc-99m labeling of theprotein occurs probably by binding to native free sulfhydryl groups ofthe protein. However, considerable amounts of Tc-99m-DTPA are alsoformed and must be removed before the labeled protein can be used. Thismethod lacks the first step of reducing the disulfide bonds needed toachieve high yields of strongly bonded radionuclide.

Sundrehagen, E., Patent Cooperation Treaty International PatentApplication No. WO 85/03231 (filed Jan. 18, 1985), used gentisic acid tostabilize the low concentrations of Sn (II) used to reduce thepertechnetate. This method is useful in minimizing radiochemicalimpurities such as radiocolloids and oxidized radionuclide. This methodlacks the first step of reducing the disulfide bonds needed to achievehigh yields of strongly bonded radionuclide.

Lees, R. S., U.S. Pat. No. 4,647,445, Radiolabelled Lipoproteins andMethod of Making Same uses dithionite at pH 8 to 9 to reduce bothpertechnetate and lipoproteins simultaneously. This method requires thatthe labeled product be purified by column chromatography to removeradionuclidic impurities prior to use. Breedveld, F. C., et al.,"Imaging of Inflammatory Arthritis with Technetittm-99m-Labeled IgG,"Journal of Nuclear Medicine, Vol. 30, No. 12, pp. 2017-2021, 1989, firstreduce pertechnetate with hydrochloric acid and then extract, transfer,and reduce the radionuclide to dryness. The protein is added to thevessel containing the dry, reduced radionuclide. Labeling is achievedduring a 60 minute incubation at 40° C. This method requires extensivepreprocessing of the radionuclide and is thus not readily applied to aninstant kit process for labeling and formulating a drug.

McKenzie, I., et al., "Coupling of the ^(99m) Technetium-Nitrido Groupto Monoclonal Antibody and Use of the Complexes for the Detection ofTumors in Mice," Journal of the National Cancer Institute, Vol. 77, pp.431-439, 1986, and Patent Cooperation Treaty Patent Application No. WO87/04164 (filed Jan. 6, 1987), reduce antibodies to provide freesulfhydryl group binding sites or introduce free sulfhydryl groupsbinding sites and label the sites with ^(99m) TcN(Cl)₄ --. The productrequires purification by gel chromatography to remove radiochemicalimpurities prior to use.

All previous methods are limited because they fail to provide a one-steplabeling kit and method which yields, within 15 minutes, an injectableproduct free of significant radiochemical impurities and a product inwhich the radionuclide is quantitatively and strongly bonded to theprotein without altering immunoreactivity when the protein being labeledis an antibody. In addition, many previous methods employ potentiallytoxic chemicals. Although the pretinning method of Crockford and Rhodeswas able to provide a rapid, one-step labeling process yieldingapproximately 85% of the radionuclide strongly bonded to the protein,the radiochemical purity has not been high enough for clinicalapplication of all monoclonal antibodies, and some products requirefinal purification prior to patient administration. In the presentinvention, both the optimum conditions for reducing the protein and theoptimum conditions for reducing the radionuclide can be achieved whileretaining the convenience of one-step labeling. This is achieved byadding steps to the manufacturing process. The protein is reduced withSn (II) as in the original pretinning method described by Crockford andRhodes. After reduction is completed, the reduced protein is put througha purification or complexing step to remove excess reducing agent andreaction by-products such as stannic chloride or other Sn (IV) agents.Alternately, an organic reducing agent is used to reduce the protein,the organic reducing agent and reaction products then removed, and Sn(II) is added to form a Sn (II) and sulfur containing protein complex. ASn (II) radionuclide reducing agent is added to the reduced proteinsolution in concentrations and with complexing agents which are optimumfor subsequent radiolabeling. The reduced protein together with theradionuclide reducing solution are aliquoted, frozen and optionallylyophilized for storage until needed for radiolabeling.

Another alternative is to reduce the antibody using the pretinningmethod of Crockford and Rhodes disclosed in the '200 patent at optimalconcentration of reagents for reducing the protein and forming the Sn(II) and sulfur containing protein complex, and then diluting thissolution with reagents to achieve conditions which are optimal forreducing the radionuclide and causing it to transfer to the protein.

SUMMARY OF THE INVENTION DISCLOSURE OF THE INVENTION

In accordance with the present invention, a method is provided forradiolabeling proteins with technetium or rhenium in which a reducingagent is used to reduce the disulfide bonds in the protein; excessreducing agent, reaction by-products and any impurities are removed;and, an optimum low concentration and limited mount of pertechnetate orperrhenate reducing agent added to reduce the sodium pertechnetate orperrhenate and facilitate the rapid labeling of the reduced protein byligand exchange.

In the preferred embodiment, a protein substrate to be radiolabeled isadmixed with a solution of Sn (II) chloride composition having a pH ofbetween about 4.5 and about 8.5, and preferably about pH 5.6, thesolution further comprising a mixture of sodium potassium tartrate andpotassium hydrogen phthalate, the pH adjusted to approximately 5.6±0.05using sodium hydroxide, and the resulting solution purged of oxygen.Alternatively, the solution may include other salts such as sodiumchloride, sodium acetate, gentisic acid, or stannous fluoride. The Sn(II) salt solution is added to the protein in an oxygen freeenvironment, and the protein and Sn (II) salt solution allowed toincubate for several hours (usually twenty-one hours) in the absence ofoxygen and at room temperature. Alternately higher or lower incubationtemperatures may be used with a corresponding inverse change in theincubation time, such that if the incubation temperature is increased,the incubation time is decreased, and vice versa. For certain proteins,such as some monoclonal antibodies or fragments thereof, the reactiontime may be shortened to less than 21 hours to prevent excessivefragmentation of the antibody protein, and can be further reduced if theprotein already contains reactive sulfide groups.

Alternatively, the protein may be reduced more rapdly by incubating itwith a disulfide group reducing agent, such as dithiothreitol (DTT) inthe ratio of 10 mg of DTT per 5 mg of protein per ml of aqueous solutionat pH 7.5 to 9.0 for 30 minutes at room temperature. The reducing agentis removed, by purification by any means. This is followed by addingdilute HCl to reduce the pH to 5.6±0.05, and then adding Sn (II)tartrate at pH 5.6±0.05.

Following incubation, the protein and Sn (II) salt solution is eitherfrozen to stop the reduction reaction, or is immediately purified bysize exclusion chromatography using an appropriate gel in a columnequilibrated with saline. The protein and buffered Sn (II) salt solulionis loaded into the column, and eluted using saline, with the molecularweights of the eluant monitored and relevant fractions collected. Ifnecessary, a small amount of Sn (II) solution is added. The fractionscorresponding to the protein to be radiolabeled are collected andpooled, and concentrated by ultrafiltration. Alternatively, the proteinmay be purified by any other suitable method including such methods asdialysis, ultrafiltration, precipitation, preparative high performanceliquid chromatography, affinity chromatography, other forms ofchromatography or preparative isoelectric focusing. The resultingprotein, substantially free of excess Sn (II) salt solution, Sn (IV)salts, contaminants or proteins of molecular weight other than theprotein to be radiolabeled, can then be frozen in an oxygen free vial.

To the oxygen free vial containing the frozen purified, reduced and Sn(II) complexed protein, a solution capable of reducing sodiumpertechnetate or perrhenate in saline solution is added in a manner toprevent immediate admixing of the two solution. A pure tin pellet canalso be added to each vial. The resulting combination is prepared aslayers of frozen solutions or is otherwise prepared without allowing anyreaction between the frozen, purified, and reduced protein and thesolution for reducing the sodium pertechnetate or perrhenate. A carrierprotein may also be added to protect against radiolysis of the purifiedand reduced protein, and to prevent adhesion of the purified and reducedprotein to surfaces, such as the vial wall. A layer of carrier protein,such as non-reduced human serum albumin or another inert diluent such asinositol or another inert sugar, or an amino acid, such as glycine, isadded, and the layer is frozen or otherwise prepared without allowingany admixture with the other solutions until use. Oxygen is excludedfrom the vial containing the two unmixed solutions. The vial is storedfrozen or it is lyophilized and stored for subsequent reconstitutionwhen radiolabeling is desired. The solution for reducing the sodiumpertechnetate or perrhenate comprises stannous chloride and a mixture ofsodium potassium tartrate and potassium hydrogen phthalate, the pHadjusted to approximately 5.6±0.05 using sodium hydroxide, and theresulting solution purged of oxygen. In practice, frequently the same Sn(II) salt solution can be used to reduce both the protein and the sodiumpertechnetate or perrhenate; however, the amount or concentration of Sn(II) salts used to reduce sodium pertechnetate or perrhenate issubstantially less than the amount of concentration used to reduce theprotein. Alternatively, the solution used for reducing sodiumpertechnetate or perrhenate can be composed of any substance whicheffectively reduces sodium pertechnetate or perrhenate and does notalter the protein to be radiolabeled, such as stannous tanrate, stannousphosphonate, stannous gluconate, stannous glucoheptonate, or othersubstances capable of reducing pertechnetate or perrhenate. The stannouschloride and all such other stannous compounds are referred to in thespecification and claims as Sn (II). No more of the pertechnetate orperrhenate reducing solution than is required to reduce the sodiumpertechnetate or perrhenate is used. This is done to prevent possibledegradation of the protein, primarily by further cleavage of disulfidebonds due to the action of the radionuclide reducing reagent.

Solid, highly pure metallic tin may be added to the vial, generally ator after freezing, and in the form of a non-oxidized tin pellet. Theaddition of metallic tin prevents oxidation loss during storage andreconstitution.

The resulting frozen or lyophilized combination of purified, reduced, Sn(II) and sulfur containing completed protein and the radionuclidereducing solution, together with the tin pellet, carrier protein andother inert diluents, are admixed with sodium pertechnetate-Tc-99m orperrhenate solution while avoiding the introduction of oxygen. Theadmixture is then incubated for a period (usually fifteen minutes) atroom temperature to allow for the reduction of the technetium or rheniumand its binding to the reduced and Sn (II) complexed protein. Theadmixture may be stabilized by the addition of human serum albumin orother similar protein in normal saline, if a carrier protein was notincluded in the original vial.

This thus provides a method for radiolabeling proteins containingreactive sulfide groups with radionuclides of technetium or rhenium toobtain stable labeling, by incubating the protein with a first reducingagent to partially reduce the disulfide bonds, purifying the reduced andSn (II) complexed protein to remove excess first reducing agent sad allimpurities, and adding only so much of second reducing agent as isnecessary to reduce pertechnetate or perrhenate. A preferred firstreducing agent is a source of Sn (II) in a solution composed of amixture of an alkali metal biphthalate and an alkali metal tartratehaving a pH of between about 5.0 and 6.0. The first reducing agent mayalso be 2-mercaptoethanol, 1,4-dithiothreitol,2,3-dihydroxybutane-1,4-dithiol, 2-aminoethanethiol HCl,2-mercaptoethylamine, thioglycolate, cyanide, cysteine or othersubstances capable of reducing disulfide bonds, with Sn (II) then addedto form an intermediate Sn (II) and sulfur containing complex. Apreferred second reducing agent is a source of Sn (II) in a solutioncomposed of a mixture of an alkali metal biphthalate and an alkali metaltartrate having a pH of preferably between about 5.0 and 6.0. The secondreducing agent may also be Sn (II) tanrate, Sn (II) gluconate, Sn (II)glucoheptonate, Sn (II) phosphonate, dithionite or other substancescapable of reducing pertechnetate or perrhenate. Following thepurification of the protein combination, the purified proteincombination can be frozen, the second reducing agent added, and thesecond reducing agent immediately frozen so that no chemical reactionoccurs between the purified protein combination and second reducingagent prior to thawing for use. It is also possible, following thefreezing of the protein combination and the second reducing agent, tolyophilize the composition. At or subsequent to the addition of thesecond reducing agent, solid, non-oxidized metallic tin can be added tothe combination of the protein combination and the second reducingagent. Purification may be accomplished by passage of the proteincombination through a size exclusion chromatography column, or bymethods such as use of a desalting column, dialysis, ultrafiltration,precipitation, preparative high performance liquid chromatography,affinity chromatography, or preparative isoelectric focusing. Optimalresults are obtained when the concentration of the protein in theprotein combination and second reducing agent is at least 1 milligramper milliliter of solution, and the volume of the protein combinationand second reducing agent is at least 2 milliliters.

This invention provides for a composition suitable for use in preparinga protein having a stable label of a radionuclide of technetium orrhenium, which composition comprises a protein which has been reduced sothat a radionuclide and sulfur containing complex can be formed, so muchof a Sn (II)-containing reducing compound for pertechnetate orperthchute as will reduce the pertechnetate or perrhenate withoutfurther reduction of the protein, and optionally pure, non-oxidizedmetallic tin. The source of pure, non-oxidized metallic tin may be a tinpellet. The composition may be made using a reduced antibody or antibodyfragment as the reduced protein. An inert career substance may also beadded to the composition, such as an inert sugar or non-reduced inertprotein. The composition may be lyophilized, preferably buffered at a pHof 4 to 6.

A kit is also provided which includes the frozen or lyophilizedcombination of purified and Sn (II) and sulfur containing complexedprotein and the radionuclide reducing solution in a single oxygen purgedvial, together with stabilizing agents, if required, ready forradiolabeling.

Proteins with either monosulfide or disulfide bonds can be radiolabeledwith radionuclides such as technetium or rhenium by incubation with afirst Sn (II) agent, or other reducing agent, which may be as describedabove. The period of incubation must be sufficient to allow formation ofSn (II) and sulfur containing complexes. As a result of the formation ofthe complexes, Sn (IV) reaction by-products, such as stannic chloride,and other impurities, such as protein fragments or polymers, may beformed. A purification step is then employed to substantially remove Sn(IV) and other impurities. A second Sn (II) agent may then be added tothe protein containing Sn (II) and sulfur containing complexes, in anamount sufficient to reduce the radionuclide. The radiolabeling is thenaccomplished by adding the radiolabel, whereby the second Sn (II) agentreduces the radionuclide and the reduced radionuclide forms radionuclideand sulfur containing complexes in the protein.

Radiolabeling may also be accomplished by omitting the step of additionof a second Sn (II) agent, in which case the residual first Sn (II)agent reduces the radionuclide and the reduced radionuclide formsradionuclide and sulfur containing complexes in the protein. This may beaccomplished by diluting the reaction mixture after the proteincontaining Sn (II) and sulfur containing complexes have been formed.

These methods are particularly applicable to technetium-99m in the formof sodium pertechnetate. Both the first and second Sn (II) agents areoptimally present in a solution containing alkali metal tartrate at a pHof between 5.0 and 6.0. The second Sn (II) agent can also consist ofsubstances such as stannous glucoheptonate, stannous gluconate, stannousphosphonate, dithionate, or other substances capable of reducingradionuclides.

These methods are particularly applicable to monoclonal antibodies,monoclonal antibody fragments and polyclonal antibodies. These methodscan be used to make product, which product on radiolabeling withtechnetium or rhenium by introduction of sodium pertechnetate orperrhenate and an incubation period, is further characterized by having85% or more of the technetium or rhenium strongly bonded to the protein.Further, when product is made using monoclonal antibodies or monoclonalantibody fragments, the immunoreactivity of the product is substantiallythe same as the immunoreactivity of the antibody prior to incubationwith the first Sn (II) agent. The product may be lyophilized, and doesnot require filtration or purification prior to patient in vivoadministration.

Proteins with either monosulfide or disulfide bonds may also beradiolabeled with radionuclides such as technetium or rhenium using amodification of the method above. The protein is incubated with a firstSn (II) agent, which may be as described above. The period of incubationmust be sufficient to allow formation of Sn (II) and sulfur containingcomplexes. As a result of the formation of the complexes, Sn (IV)reaction by-products and other impurities may be formed. Sn (IV)reaction by-products can then be complexed with a polyaminocarboxylicacid, such as ethylenediaminetetraacetic acid (EDTA) ordiethylenetriaminopentaacetic acid (DTPA). Radiolabeling is accomplishedby adding the radionuclide, whereby the residual first Sn (II) agentreduces the radionuclide and the reduced radionuclide forms radionuclideand sulfur containing complexes in the protein.

Radiolabeling may optimally be performed by the addition of a second Sn(II) agent, to facilitate reduction of the radionuclide,polyaminocarboxylic acid.

The methods using polyaminocarboxylic acid are particularly applicableto technetium-99m in the form of sodium pertechnetate. Both the firstand second Sn (II) agents are optimally present in a solution containingalkali metal tartrate at a pH of between 5.0 and 6.0. The second Sn (II)agent can also consist of substances such as stannous glucoheptonate,stannous gluconate, stannous phosphonate, or other substances capable ofreducing radionuclides.

The methods using polyaminocarboxylic acid are particularly applicableto monoclonal antibodies, monoclonal antibody fragments and polyclonalantibodies. These methods can be used to make product, which product onradiolabeling with technetium by introduction of sodium pertechnetateand an incubation period, is further characterized by having 85% or moreof the technetium strongly bonded to the protein. Further, when productis made using monoclonal antibodies of monoclonal antibody fragments,the immunoreactivity of the product is substantially the same as theimmunoreactivity of the antibody prior to incubation with the first Sn(II) agent. The product may be lyophilized, and does not requirefiltration or purification prior to patient in vivo administration.

All the foregoing methods result in a proteinaceous composition in whichis found a Sn (II) and sulfur containing complex, comprising reactivesulfide groups complexed with Sn (II), and which composition is suitablefor radiolabeling with radionuclides such as technetium and rhenium. Thecomposition may also comprise a Sn (II) reducing agent in an mountsufficient to reduce the radionuclide, and may also contain a completingagent, preferably a polyaminocarboxylic acid such as EDTA or DTPA. Theprotein may be a monoclonal antibody, monoclonal antibody fragment orpolyclonal antibody, and may be made into a product. The product may belyophilized. On radiolabeling with technetium by introduction of sodiumpertchnetate and an incubation period, the product is furthercharacterized by having 85% or more the technetium strongly bonded tothe protein. It is also further characterized by requiring an incubationperiod of fifteen minutes or less.

Accordingly, it is an object of the present invention to provide amethod for direct labeling of proteins with technetium or rhenium, whichmethod will eliminate undesirably fragments or otherwise degradedprotein components from the final product.

It is a further object of the present invention to provide a methodwhich results in increased radiolabeling efficiencies utilizingtechnetium or rhenium as the radioisotope.

It is a further object of the present invention to provide a method toradiolabel antibodies or antibody fragments without loss of affinity ofthe antibodies or antibody fragments due to the radiolabeling process.

It is a further object of the present invention to provide a method forradiolabeling proteins with technetium or rhenium which does not usepotentially toxic or injurious chemicals or substances in the method.

Another object of the present invention is to provide a method and kitwhich permits radiolabeling to be accomplished by the end user using asingle vial, containing both reduced antibody and stannous ions, andfurther containing a means to maintain low quantities of stannous ionswhile protecting against oxidation loss, which method requires only asingle step to accomplish radiolabeling, being the introduction ofsodium pertechnetate.

Other objects, advantages and novel features, and further scope ofapplicability of the present invention will be set forth in part in thedetailed description to follow and in part will become apparent to thoseskilled in the art upon examination of the following, or may be learnedby practice of the invention. The objects and advantages of theinvention may be realized and attained by means of the instrumentalitiesand combinations particularly pointed out in the appended claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION (BESTMODES FOR CARRYING OUT THE INVENTION)

Any protein, chelating agent or other substrate which contains one ormore monosulfide or disulfide bonds which can be reduced can beradiolabeled in accordance with this invention. Representative suitablesubstrates include human serum albumin, fibrinogen, urokinase, gammaglobulin, glycoproteins, other proteins and antibodies, or antibodyfragments, of any species, and including both polyclonal and monoclonalantibodies made by any means, as well as chimeric and geneticallyengineered antibodies, and antibody fragments of all of the foregoing.This includes immunoglobulins of any class, such as IgG, IgM, IgA, IgD,or IgE, of any species origin, including human beings, chimericantibodies or or hybrid antibodies with dual or multiple antigen orepitope specifities, and fragments of all the foregoing, includingF(ab')₂, F(ab)₂, Fab', Fab and other fragments, including hybridfragments, and further includes any immunoglobulin or any natural,synthetic or genetically engineered protein that functionally acts likean antibody by binding to a specific antigen to form a complex. The term"antibody" or "antibodies", and the phrase "monoclonal antibodycomponent", as used throughout the specification and claims is intendedto include all such antibodies and antibody fragments.

The present invention, through inclusion of a purification step, andconcomitant removal of excess reducing reagents, presents a number ofsignificant advantages. By removal of all species of reduced proteinother than the protein to be radiolabeled, including smaller or largermolecular weight species, competition for reduced Tc-99m is eliminated.This results in significantly higher radiolabeling yields. By keepingthe total amount of stannous and stannic ions in the pertechnetate orperrhenate reducing solution as low as possible, the formation ofadditional reduced protein species is minimized. It generally takes farfewer stannous ions to reduce pertechnetate or perrhenate than to reducethe disulfide bonds in proteins, thereby allowing reduction of disulfidebonds in the protein to be radiolabeled, which additional reductioncould result in protein species other than the protein to beradiolabeled.

The present invention also presents significant advantages because itrequires only one step to accomplish radiolabeling by the end user, theaddition of sodium, pertechnetate or perrhenate and the concomitantincubation thereof. This significant simplification is possible becauseboth the stannous ions and the reduced antibody are frozen, togetherwith a carrier protein and other inert diluents, and optionallylyophilized, in the same vial. The addition of the tin pellet, or othersource of purified and non-oxidized metallic tin, further preserves thelow concentration of stannous ions and helps prevent loss of radiolabeldue to oxidation dung storage or reconstitution.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE I

A Sn (II) reducing solution was made composed of stannous chloride in atartrate-phthalate solution. Stannous chloride was prepared bydissolving stannous chloride crystals in concentrated hydrochloric acidat 0.5M, yielding approximately 94.8 mg of stannous chloride per ml.This solution was then stored in sealed and nitrogen purged vials untilused. Alternate sources for stannous chloride can be used, including useof a non-oxidized solid tin pellet contacted with concentratedhydrochloric acid.

A tartrate-phthalate solution of a mixture of sodium potassium tartrateand potassium hydrogen phthalate was prepared. 0.282 g of sodiumpotassium tartrate was dissolved in 100 ml of distilled water, and tothis was added 0.817 g potassium hydrogen phthalate. The pH was adjustedto approximately 5.0, using 10N sodium hydroxide, and was then adjustedto 5.6±0.05 using 1N sodium hydroxide. The resulting solution wasstirred and purged of oxygen by bubbling an inert gas, such as nitrogen,helium or the like, through the substance. The Sn (II) reducing solutionwas made by measuring a portion of the purged tartrate-phthalatesolution into a flask, and slowly adding thereto a volume of thestannous chloride, It is preferable if a stir bar or similar mechanismis used to insure mixture of the stannous chloride as it is added to thesolution. Approximately one volume of stannous chloride to 100 volumesof buffer was used. The pH was continuously monitored, and once thestannous chloride was added, the pH was adjusted to approximately 5.0,using 10N sodium hydroxide, and was then adjusted to 5.6±0.05 using 1Nsodium hydroxide.

All steps are undertaken in an oxygen free environment, and may be donewhile bubbling an inert gas through the solution. The resulting solutionis purged of oxygen by bubbling an inert gas, such as nitrogen, heliumor the like, through the substance.

Alternately, the antibody reducing solution can be made using otherdisulfide reducing agents such as 2-mercaptoethanol 1,4 dithiothreitol,2,3 dihydroxybutane-1,4-dithiol, 2-aminoethanethiol HCl,2-mercaptoethylamine, thioglycolate, cyanide, cysteine, or otherdisulfide splitting reagents.

The protein to be reduced is placed in a nitrogen purged, sealed vial.If monoclonal antibody or fragments thereof are to be labeled, of aminimum of 0.1 mg, and preferably at least 2 mg, of monoclonal antibodyor fragments is used, at a concentration of 1 mg or more per ml,preferably 1.7 mg/ml. The monoclonal antibody, fragments or otherprotein may be diluted in normal saline, or concentrated byultrafiltration, as may be necessary. To the protein, Sn (II) reducingsolution is added, in the approximate ratio of 3 volumes of protein to 2volumes of Sn (II) reducing solution. The vial containing the proteinand Sn (II) reducing solution is then allowed to incubate, preferably inan oxygen free environment, such as a container filled with nitrogen oranother inert gas. The vial is allowed to incubate at room temperaturefor a period sufficient to allow reduction of the disulfide bonds in theprotein. Generally, incubation for between one and twenty-four hours atroom temperature is adequate, with incubation for twenty-one hours atroom temperature being preferred for most whole immunoglobulins. If thetemperature is increased, the incubation time is decresed, andconversely, incubation at lower temperatures requires correspondinglylonger incubation times. Incubation is terminated by freezing theadmixture of protein and Sn (II) reducing solution by dilution or byproceeding immediately to purification.

When other reducing agents are used, such as 2-metcaptoethanol,1,4-dithiothreitol, 2,3-dihydroxybutane-1,4-dithiol, 2-aminoethanethiolHCl, 2-mercaptoethylamine, thioglycolate, cyanide, cysteine, or otherdisulfide splitting reagents, the incubation time must be adjusted forthe specific reducing agent being used. Generally, a shorter incubationtime is required. After the reducing solution is removed, Sn (II) saltis added to form a Sn (II) and sulfur containing complex, and thuspreserve the reduced disulfide groups during subsequent purificationsteps. The purification steps undertaken with these reducing agents mustremove substantially all of the reducing agent.

The admixture of protein and Sn (II) reducing or other reducing solutionis purified, preferably by size exclusion chromatography. A column ispacked with an appropriate gel, such as Sephacryl-200 gel (Pharmacia,Piscataway, N.J.) for use with low molecular weight monoclonal antibodyF(ab')₂ fragments. The column is equilibrated with an appropriateelution buffer, such as gentisate buffered saline. The admixture ofprotein and reducing solution is applied to the column and fractionated,using the elution buffer. A fraction profile is generated using anultraviolet detector or similar device, and the fractions containing theresulting protein to be radiolabeled are collected and pooled. Theresulting reduced protein is concentrated, if necessary, preferably to aconcentration of 1.7 mg/ml. Concentration can be accomplished byultrafiltration. The resulting concentrate can also be dialyzed againstnormal saline to remove any residual Sn (IV) salts or other residualreducing agent.

Alternately, the admixture of Sn (II) and sulfur conraining complexedprotein and Sn (II) or other reducing solution may be purified bypassage of the admixture through a desalting column, collecting thereduced protein and discarding the Sn (IV) salts and excess reducingreagents. The resulting eluant can be concentrated and dialyzed asrequired. This method will remove excess Sn (II) solution. Sn (IV)reaction by-product and other reducing reagents, but will notnecessarily remove other impurities. such as smaller fragments of theprotein resulting from over reduction of the disulfide bonds in theprotein or aggregation of reduced proteins. These small fragments orlarge aggregates, in the case of monoclonal antibodies, may not beimmunoreactive, or may have a biodistribution different than that of thedesired protein, necessitating their removal from the final product.

Alternately, the admixture of reduced protein and Sn (II) or otherreducing solution may also be purified by other means, includingdialysis, ultrafiltration, precipitation, preparative high performanceliquid chromatography, affinity chromatography, other forms ofchromatography, preparative isoelectric focusing, and other purificationmeans common to the art.

The purified, reduced and Sn (II) complexed protein is then purged ofoxygen, preferably by bubbling an inert gas such as nitrogen through thesolution, and can be frozen in oxygen-purged vials, To each vialcontaining frozen protein solution, pertechnetate reducing solution isadded, and the contents either immediately frozen or lyophilized, sothat no reaction takes place between the frozen protein solution and thepertechnetate reducing solution. A non-oxidized tin pellet may also beadded to the vial, which tin pellet will replace trace mounts of Sn (II)and help to stabilize the low concentration of stannous ions in thepertechnetate reducing solution, and to help prevent losses ofradiolabel due to reoxidation dunng storage of the radiolabeled product.The pertechnetate reducing solution can be made as the Sn (II) reducingsolution was made, except that approximately one volume of stannouschloride at 0.5M is added to approximately 5,000 volumes oftartrate-phthalate solution, resulting in a pertechnetate reducingsolution of approximately 0.1 millimolar. Approximately one volume ofpertechnetate reducing solution is added to two volumes of purified,reduced and Sn (II) complexed protein. Optimally, the protein solutionis at a concentration of approximately 1.7 mg/ml, and to each 1.32 ml ofthe purified, reduced and Sn (II) complexed protein solution,approximately 0.68 ml of pertechnetate reducing solution is added.

Alternately, the pertechnetate reducing solution can be made using Sn(II) glucoheptonate, Sn (II) tartrate, Sn (II) phosphonate, dithioniteand other commonly used Tc-99m radiopharmaceutical kits.

The pertechnetate reducing solution can also be used to reduceperrhenate. The discussion of pertechnetate and pertechnetate compoundsthroughout the specification is also applicable to perrhenate andperrhenate compounds. Likewise, the discussion throughout thespecification of technetium and its compounds is appliable to rheniumand its compounds.

To radiolabel, the desired activity of sodium pertechnetate-Tc-99m isadded and admixed, and the admixture allowed to incubate for a period,generally approximately fifteen minutes. This step is conducted whileavoiding or minimizing the introduction of atmospheric oxygen. Ifdesired, the resultant radiolabeled protein can be stabilized by theaddition of 1% human serum albumin in normal saline or other suitableprotective protein.

The source of technetium-99m is conventionally obtained as sodiumpertechnetate-Tc-99m from a 99Mo/99mTc generator. Any source ofpharmaceutically acceptable technetium-99m may be utilized in thepresent invention.

Alternatively other radioisotopes of technetium and isotopes of rheniumsuch as Re-186 and Re-188 may be used. When perrhenate rather thanpertechnetate is reduced, usually a higher temperature and a longerradiolabeling time are required to carry the reaction to completion.

EXAMPLE II

This example illustrates the process of this invention for labelingimmunoglobulin G (IgG). IgG is obtained from animals such as sheep,goats, mice or humans. Sodium Pertechnetate-Tc-99m U.S.P. is obtainedfrom any commercial source.

A Sn (II) disulfide bond reducing agent was prepared by adding 0.2 ml of0.5M stannous chloride in concentrated HCl (12M) to 20 ml of 40 mMpotassium biphthalate and 10 mM sodium tartrate solution (pH of 5.6).The stannous chloride was prepared by adding the concentratedhydrochloric acid to non-oxidized pellets of SnCl₂ having a surface freeof dull stannous oxide. The pH of the resultant reducing solution thenwas brought up to 5.6±0.05, by adding 10M NaOH to a pH of 5.5, andadding 1M NaOH to adjust to the final pH.

An IgG preparation was made by diluting 0.25 ml of Immune Globulin(Human), U.S.P., Cutter Biological, which contained 15-18% proteinstabilized with 0.21-0.32M glycine, with 7.25 ml of Sterile Water forInjection, U.S.P., and filtering through a 0.22 micron filter. 5 ml ofthe Sn (II) reducing solution was mixed with 7.5 ml of the IgGpreparation. The vial containing the admixed solutions was sealed andflushed with N₂ gas to remove oxygen. This admixed solution was storedfor 21 hours at room temperature in the dark to allow for the partialreduction of disulfide bonds to form what is referred to subsequently asreduced protein. After the 21 hour incubation the contents of the vialwas passed through a PD-10 desalting column (Pharmacia LKBBiotechnology, Piscataway, N.J.); the protein containing fraction wascollected and the remaining eluate, which contained the Sn (II), Sn (IV)and other salts, was discarded. The reduced and Sn (II) complexedprotein fraction was concentrated by ultrafiltration to a concentrationof 1.7 mg/ml. 0.5 mg aliquots of reduced and Sn (II) complexed proteinwere placed in sealed, N₂ gas filled serum vials and frozen. A Sn (II)pertechnetate reducing solution was made of 0.5 ml of 0.1 mM SnCl₂ in 40mM potassium biphthalate/10 mM sodium tartrate at a pH of 5.6. The Sn(II) pertechnetate reducing solution was added without allowing thereduced antibody solution to thaw, and this solution was also frozen. Asterile, 3 mm diameter tin metal shot was added, the vial flushed withN₂ and stored at -20° C. until needed for radiolabeling.

To radiolabel the gamma giobulin preparation with Tc-99m, 1.0 ml ofSodium Pertechnetate-Tc-99m, U.S.P., containing 2.5 mCi ofradioactivity, was added to the vial, and the vial and contents broughtto room temperature, mixed and allowed to stand for 15 minutes. Thinlayer chromatographic analysis of the product revealed that 99.6% of theradioactivity was protein bound. High performance liquid chromatography,using both UV and radioisotope detectors, showed that the Tc-99m elutionparalleled the protein elution profile.

EXAMPLE III

This example illustrates the process of this invention for labelingmonoclonal murine antibodies of IgG and IgM classes. The antibody wasobtained from murine ascites or bioreactor fluid, purified to greaterthan 95%, and prepared at concentrations of greater than 1 mg/ml in 0.9%NaCl solution.

A Sn (II) reducing solution was prepared as in Example II. Two wholeantibody preparations were tested; B72.3, an IgG murine antibody, andanti-SSEA-1, an IgM murine antibody. Each antibody preparation was at aprotein concentration of 1.7 mg/ml. To each ml of purified proteinsolution was added 0.66 ml of Sn (II) reducing solution. The admixedsolutions were incubated and passed through a PD-10 column as in ExampleII. The reduced protein fraction was concentrated by ultrafiltration toa concentration of 2 mg/ml. Aliquots of reduced protein, containing from0.5 to 2.0 mg protein, were placed in sealed, N₂ gas filled serum vialsand frozen.

A pertechnetate reducing solution was prepared by dissolving 50 mg ofgentisic acid, 0.375 μg SnCl₂ and 975 μg of sodium potassium tartrate in50 ml of distilled water which had previously been deoxygenated bybubbling N₂ gas through it for two to three minutes. The pH was adjustedto 7.0 by addition of very dilute (0.05N) NaOH. Equal volumes of thissolution were layered over the frozen, reduced and Sn (II) complexedprotein solution and this solution frozen. A sterile, 3 mm diameter tinmetal shot was added, the vial flushed with N₂ and stored at -20° C.until needed for radiolabeling.

To radiolabel the IgG or IgM preparations with Tc-99m. 1.0 ml of SodiumPertechnetate-Tc-99m, U.S.P., containing 2.5 mCi of radioactivity, wasadded to each vial, and the vial and contents brought to roomtemperature, mixed and allowed to stand for 15 minutes. Thin layerchromatographic analysis of the products revealed that 90.0% to 96.5% ofthe radioactivity was protein bound. High performance liquidchromatography (HPLC), using both UV and radioisotope detectors, showedthat the Tc-99m elution paralleled the protein elution profile. Nonon-protein bound radioactivity was found by HPLC analysis.

EXAMPLE IV

This example illustrates the process of this invention for labelingF(ab')₂ fragment of monoclonal antibody. This example also shows thatthe composition of the radiolabeled product varies with the method andtype of disulfide reducing reagent used. This example also shows thatthe current method is superior to the original direct labeling method ofCrockford and Rhodes, U.S. Pat. No. 4,424,200 entitled "METHOD FORRADIOLABELING PROTEINS WITH TECHNETIUM-99M", for this particularmonoclonal antibody fragment. The F(ab')₂ fragment was obtained bypepsin digestion of murine monoclonal antibody followed bychromatographic purification which separated the F(ab')₂ fragments fromother material found in the pepsin digest with greater than 95% purity.

The monoclonal antibody fragment used in this example was obtained fromSorin Biotechnica, Italy. It was a murine anti-CEA F(ab')₂ whichprevious experimentation had shown very poor radiolabeling with Tc-99musing the pretinning method.

Four different radiolabeling procedures were employed; one used theoriginal pretinning method described in U.S. Pat. No. 4,424,200, and theother three procedures used methods taught in this invention. The fourprocedures can be summarized as follows:

1. The original pretinning method described in U.S. Pat. No. 4,424,200,in which a Sn (II) reducing solution was prepared as described inExample II, but no purification step was employed, and no pertechnetatereducing solution was added.

2. The method of this invention using Sn (II) reducing solution toreduce disulfide bonds in the antibody fragment, as described in ExampleII, including a purification step using a PD-10 desalting column, and apertechnetate reducing solution composed of stannous salt with a tinpellet.

3. The method of this invention using 2-mercaptoethanol to reducedisulfide bonds in the antibody fragment. A 5% solution of2-mercaptoethanol was prepared in 0.1M phosphate buffer at pH 8.0. Oneml of this solution was added to 1 mg of the lyophilized antibodyfragment protein and mixed to dissolve. After 1 hour incubation at roomtemperature, 1.6 ml of saline was added and the partially reducedprotein separated from the other components in the solution by passagethrough a PD-10 desalting column. The protein was concentrated byultrafiltration to 1.7 mg/ml. A pertechnetate reducing solution composedof stannous salt with a tin pellet, as described in Example II, was thenapplied.

4. The method of this invention using dithiothreitol to reduce disulfidebonds in the antibody fragment. 15.4 mg of dl-dithiothreitol (DTT) wasdissolved in 10 ml of a solution of 50 mM tris and 1 mM EDTA at pH 8.0.For each mg of lyophilized protein to be reduced, 0.33 ml of thereducing solution was added with mixing to dissolve the protein. Thereduction mixture was allowed to react at 37° C. for one hour. Thepartially reduced protein was purified by size exclusion columnchromatography with collection of the protein fraction corresponding tothe molecular weight of the original F(ab')₂ antibody fragment. Thechromatographically purified fragment was concentrated to 1.7 mg/ml in0.9% saline by ultrafiltration. A pertechnetate reducing solutioncomposed of stannous salt with a tin pellet, as described in Example II,was then applied.

For each of the four preparations, the frozen and vialed antibodyfragment was radiolabeled and tested as described in Example II. Theresults of the four different procedures utilizing the same antibodyfragment are listed in Table 1.

Table 1 shows that this specific F(ab')₂ murine monoclonal antibodyfragment effectively fails to radiolabel using the original pretinningmethod taught in U.S. Pat. No. 4,424,200, in which there is nopurification step and no addition of limited mounts of pertechnetatereducing solution, yet effectively radiolabels using methods of thisinvention, particularly when an agent other than Sn (II) was used toreduce the disulfide bonds. The same pertechnetate reducing agent, hereSn (II), was used in each procedure. Conditions were not optimizedduring the procedure to obtain greater than 85% binding. The purpose ofthe comparison was to show how the binding can be grossly improved byusing the process of invention.

                                      TABLE I                                     __________________________________________________________________________    COMPARISON OF RESULTS USING DIFFERENT METHODS                                 FOR PARTIAL REDUCTION OF THE DISULFIDE                                        BONDS OF ANTI-CEA F(ab').sub.2                                                Disulfide Reduction Method                                                                      Results                                                     __________________________________________________________________________      Original pretinning U.S. Pat. No.                                                             4-9% of the Tc-9m bound to F(ab').sub.2 ; 45-53%              4,424,200 method                                                                              bound to smaller fragments.                                   This invention using Sn (II)                                                                  18-29% of the Tc-99m bound to F(ab').sub.2 ; 51-76%                           bound to smaller fragments.                                   This invention using                                                                          79% of the Tc-99m bound to F(ab').sub.2 ; none                2-mercaptoethanol                                                                             bound to smaller fragments.                                   This invention using DTT                                                                      74% of the Tc-99m bound to F(ab').sub.2 ; none                                bound to smaller fragments                                  __________________________________________________________________________

EXAMPLE V

This example illustrates the process of this invention for labeling amonoclonal antibody which cannot be satisfactorily labeled by theoriginal direct or pretinning method, or by other equivalent directlabeling methods. The reason for the failure of the previous directmethods with certain monoclonal antibodies is that during the reductionof the antibody either fragmentation or aggregation of the antibodyoccurs which results in protein species of altered molecular weight. Anexample of this is an anti-CEA murine monoclonal IgG provided by SorinBiomedia, Italy. When this antibody is reduced with Sn (II) salts, smallmounts of fragments are formed which label preferentially with thereduced Tc-99m. When this antibody is reduced with dithiothreitol or2-mercaptoethanol, dimers and polymers of reduced IgG are formed whichlabel with Tc-99m. By the method of this invention, the antibody, afterthe disulfide bond reduction step, is purified by passage through a sizeexclusion chromatograph column. The column eluate corresponding only tothe molecular weight of the original antibody was separated from boththe smaller or larger protein species. A quantity of pertechnetatereducing solution sufficient to reduce the sodium pertechnetate but notto further reduce disulfide bonds in the antibody was added, and theantibody radiolabeled. The resulting Tc-99m labeled protein was of thecorrect molecular weight and free of the smaller or large molecularweight contaminants.

EXAMPLE VI

This example illustrates the use of purification and EDTA to obtainhigher HPLC yields.

Four aliquots of a chimeric IgG monoclonal antibody were reduced with aSn (II) reducing agent as in Example II. Two aliquots were purified bypassage through a PD-10 column, followed by addition of Sn (II)pertechnetate reducing solution. The remaining two aliquots were notpurified, and no additional Sn (II) pertechnetate reducing solution wasadded. To one of each of the two pairs of aliquots,ethylenediaminetetraacetic acid (EDTA) was added prior to radiolabeling.

Quantitative radiochemical purity was determined by HPLC yield. In thismethod, the percent of strongly bonded Tc-99m is determined as thepercent of Tc-99m recovered in HPLC size exclusion chromatography of thetotal Tc-99m injected multiplied by the percent of Tc-99m under the IgMprotein peak. Weakly bonded Tc-99m is almost completely transferred andbound to the HPLC column, with unreduced Tc-99m elutes, but not with theprotein peak. This method thus provides a measure of strongly bondedTc-99m.

                  TABLE 2                                                         ______________________________________                                        COMPARISON OF RESULTS USING EDTA TO                                           PARTIALLY BIND IMPURITIES                                                                                 HPLC                                              Reduction Method            Yield                                             ______________________________________                                        1.  SN (II) reduction, no purification                                                                        52%                                           2.  SN (II) reduction, no purification, EDTA                                                                  78%                                           3.  SN (II) reduction, PD-10 desalting column,                                                                96%                                               SN (II) pertechnetate reducing solution                                   4.  SN (II) reduction, PD-10 desalting column,                                                                97%                                               SN (II) pertechnetate reducing solution, EDTA                             ______________________________________                                    

EXAMPLE VII

This example illustrates the formation of reactive sulfide groups onreduction of disulfide bonds by Sn (II) and by dithiothreitol (DTT).Murine monoclonal IgM antibody was labeled with I-125 using the Iodobeadmethod. Aliquots were incubated with Sn (II) reducing solution made asin Example II; the aliquots were removed at different time intervals, upto 21 hours, and passed through a PD-10 column to stop the reductionreaction. Aliquots were also incubated with DTT. Sodium chloride wasused as a control. The PD-10 columns were eluted with nitrogen purgedsaline to prevent reoxidation of reactive sulfide groups. One portion ofeach aliquot was used for measurement of free reactive sulfide groupsand the other portion was used for TC-99m labeling and the measurementof the percent of strongly bonded Tc-99m.

Reactive sulfide group formation was determined by relative binding ofthe I-125 labeled antibody to Thio Avidgel F (Bioprobe, Justin, Calif.).Thio Avidgel F binds proteins which have free sulfhydryl or reactivesulfide groups: if such groups are formed during incubation withreducing agents, then the percent antibody binding should beproportional to reactive sulfide, groups. Tc-99m labeling wasaccomplished by using a Sn (II) pertechnetate reducing solution,prepared as in Example II, to which was added human serum albumin andinositol. The percent strongly bonded Tc-99m was determined by HPLCyield as described in Example VI.

                  TABLE 3                                                         ______________________________________                                        SULFHYDRYL GROUP CORRELATION TO                                               STRONGLY BONDED Tc-99m                                                        Reducing                                                                              Incubation Time                                                                            Percent Binding                                          Agent   (Hours)      To Thiogel  HPLC Yield                                   ______________________________________                                        NaCl    NA           10%          5.1%                                        Sn (II) 1            14%         20.9%                                        SN (II) 21           23%         46.7%                                        DTT     1            62%         57.8%                                        ______________________________________                                    

EXAMPLE VIII

This example illustrates that the process of this invention, using theSn (II) reducing agent of Example II followed by passage through a PD-10column, does not affect the immunoreactivity of monoclonal antibodies.F(ab')₂ antibody specific for myosin was spiked with I-125 labeledpolyclonal immune globulin. A portion was reduced as described andpassed through a PD-10 column. Aliquots of both the reduced F(ab')₂ andunreduced F(ab')₂ were then adjusted to yield the same concentrationusing the I-125 labeled polyclonal immune globulin as the standard.ELISA was performed against purified heavy chain myosin antigen withboth aliquots using decreasing concentrations of the F(ab')₂ antibody.The affinity constants were calculated at one-half plateau maximumabsorbance, and were found to be identical.

The preceding examples can be repeated with similar success bysubstituting the generically or specifically described reactants and/oroperating conditions of this invention for those used in the precedingexamples. In particular, other proteins, chelating agents or substrateswhich contain monosulfide or disulfide bonds which can be reduced may beused in place of IgG, IgM and F(ab')₂ monoclonal antibody; otherreducing agents can be used to reduce the disulfide bonds in thesubstance to be radiolabeled; other purification methods can be used toremove the reducing agent; other pertechnetate reducing agents can beused to reduce the sodium pertechnetate; and isotopes of rhenium can beused in addition to isotopes of technetium. The foregoing are merelyillustrative, and other equivalent embodiments are possible andcontemplated.

Although the invention has been described with reference to thesepreferred embodiments, other embodiments can achieve the same results.Variations and modifications of the present invention will be obvious tothose skilled in the art and it is intended to cover in the appendedclaims all such modifications and equivalents.

What is claimed is:
 1. A method of radiolabeling a protein containingone or more monosulfide or disulfide bonds with a radionuclide to obtainstable labeling, comprising the steps of:a) incubating the proteincontaining monosulfide or disulfide bonds with a first Sn (II) agent,the period of incubation being sufficient to allow formation of Sn(II)-containing and sulfur-containing complexes and formation of Sn (IV)reaction by-products, while preventing excessive fragmentation of theprotein; b) purifying the reduced protein to substantially removeuncomplexed Sn agents and other impurities; c) adding a second Sn (II)agent to the purified protein with Sn (II)-containing andsulfur-containing complexes in a sufficient mount to reduce theradionuclide yet not generate significant radiochemical impurities, theradionuclide to be added in a subsequent step; and d) radiolabeling thepurified protein with the Sn (II)-containing and sulfur-containingcomplexes by adding the radionuclide, whereby the Sn (II) agents reducethe radionuclide and the reduced radionuclide formsradionuclide-containing and sulfur-containing complexes.
 2. The methodof claim 1 wherein the source of the first Sn (II) agent is present in asolution comprising alkali metal tartrate having a pH of betweenapproximately 5.0 and 6.0.
 3. The method of claim 1 wherein the sourceof the second Sn (II) agent is present in a solution comprising alkalimetal tartrate having a pH of between approximately 5.0 and 6.0.
 4. Themethod of claim 1 wherein the source of the second Sn (II) agentcomprises at least one member selected from the group consisting ofstannous glucoheptonate, stannous gluconate and stannous phosphonate. 5.The method of claim 1 wherein the protein comprises a member selectedfrom the group consisting of monoclonal antibodies, monoclonal antibodyfragments and polyclonal antibodies.
 6. The method of claim 1 whereinfollowing step c), and prior to step d), the purified protein with Sn(II)-containing and sulfur-containing complexes and second Sn (II) agentare frozen in a vial, whereby the frozen purified protein with Sn(II)-containing and sulfur-containing complexes and second Sn (II) agentcan be maintained for an indefinite period before radiolabeling in stepd) by the addition of the radionuclide to the vial.
 7. The method ofclaim 1 wherein following step c), and prior to step d), the purifiedprotein with Sn (II)-containing and sulfur-containing complexes andsecond Sn (II) agent are lyophilized in a vial, whereby the lyophilizedpurified protein with Sn (II)-containing and sulfur-containing complexesand second Sn (II) agent can be maintained for an indefinite periodbefore radiolabeling in step d) by the addition of the radionuclide tothe vial.
 8. The method of claim 1 wherein 85 percent or more of theradionuclide is strongly bonded to the protein.
 9. The method of claim 1wherein the radionuclide comprises a member selected from the groupconsisting of technetium and rhenium.
 10. The method of claim 9 whereinthe radionuclide is technetium-99m in the form of sodium pertechnetate.11. A method of radiolabeling a protein containing one or moremonosulfide or disulfide bonds with a radionuclide to obtain stablelabeling, comprising the steps of:a) incubating the protein containingmonosulfide or disulfide bonds with a Sn (II) agent, the period ofincubation being sufficient to allow formation of Sn (II)-containing andsulfur-containing complexes and formation of Sn (IV) reactionby-products, while preventing excessive fragmentation of the protein; b)purifying the reduced protein to remove uncomplexed Sn agents and otherimpurities yet retaining Sn (II) in a sufficient mount to reduce theradionuclide and not generate significant radiochemical impurities, theradionuclide to be added in a subsequent step; and c) radiolabeling thepurified protein with the Sn (II)-containing and sulfur-containingcomplexes by adding the radionuclide, whereby the Sn (II) agent reducesthe radionuclide and the reduced radionuclide formsradionuclide-containing and sulfur-containing complexes.
 12. The methodof claim 11 wherein the source of the first Sn (II) agent is present ina solution comprising alkali metal tartrate having a pH of betweenapproximately 5.0 and 6.0.
 13. The method of claim 11 wherein theprotein comprises a member selected from the group consisting ofmonoclonal antibodies, monoclonal antibody fragments and polyclonalantibodies.
 14. The method of claim 11 wherein following step b), andprior to step c), the purified protein with Sn (II)-containing andsulfur-containing complexes is frozen in a vial, whereby the frozenpurified protein with Sn (II)-containing and sulfur-containing complexescan be maintained for an indefinite period before radiolabeling in stepc) by the addition of the radionuclide to the vial.
 15. The method ofclaim 11 wherein following step b), and prior to step c), the purifiedprotein with Sn (II)-containing and sulfur-containing complexes islyophilized in a vial, whereby the lyophilized purified protein with Sn(II)-containing and sulfur-containing complexes can be maintained for anindefinite period before radiolabeling in step c) by the addition of theradionuclide to the vial.
 16. The method of claim 11 wherein 85 percentor more of the radionuclide is strongly bonded to the protein.
 17. Themethod of claim 11 wherein the radionuclide comprises a member selectedfrom the group consisting of technetium and rhenium.
 18. The method ofclaim 17 wherein the radionuclide is technetium-99m in the form ofsodium pertechnetate. .[.19. A method of radiolabeling a proteincontaining one or more monosulfide or disulfide bonds with aradionuclide to obtain stable labeling, comprising the steps of:a)incubating the protein containing monosulfide or disuifide bonds with afirst Sn (II) agent, the period of incubation being sufficient to allowformation of Sn (II)-containing and sulfur-containing complexes, whilepreventing excessive fragmentation of the protein; b) complexing thefree Sn agents with a complexing agent so as not to further reduce theprotein, while retaining complexed Sn agents for reducing theradionuclide, the radionuclide to be added in a subsequent step; and c)radiolabeling the protein with the Sn (II)-containing andsulfur-containing complexes by adding the radionuclide, whereby thecompleted Sn (II) agents reduce the radionuclide and the reducedradionuclide forms radionuclide-containing and sulfur-containingcomplexes..]..[.20. The method of claim 19 wherein the complexing agentis polyaminocarboxylic acid..]..[.21. The method of claim 19 wherein thepolyaminocarboxylic acid comprises at least one member selected from thegroup consisting of EDTA and DTPA..]..[.22. The method of claim 19wherein the source of the first Sn (II) agent is present in a solutioncomprising alkali metal tartrate having a pH of between approximately5.0 and 6.0..]..[.23. The method of claim 19 wherein the proteincomprises a member selected from the group consisting of monoclonalantibodies, monoclonal antibody fragments and polyclonalantibodies..]..[.24. The method of claim 19 wherein following step b),and prior to step c), the free Sn complexes and protein with Sn(II)-containing and sulfur-containing complexes are frozen in a vial,whereby the frozen free Sn complexes and protein with Sn (II)-containingand sulfur-containing complexes can be maintained for an indefiniteperiod before radiolabeling in step d) by the addition of theradionuclide to the vial..]..[.25. The method of claim 19 whereinfollowing step b), and prior to step c), the free Sn complexes andprotein with Sn (II)-containing and sulfur-containing complexes arelyophilized in a vial, whereby the lyophilized free Sn complexes andprotein with Sn (II)-containing and sulfur-containing complexes can bemaintained for an indefinite period before radiolabeling in step d) byaddition of the radionuclide to the vial..]..[.26. The method of claim19 wherein 85 percent or more of the radionuclide is strongly bonded tothe protein..]..[.27. The method of claim 19 wherein the radionuclidecomprises a member selected from the group consisting of technetium andrhenium..]..[.28. The method of claim 27 wherein the radionuclide istechnetium-99m in the form of sodium pertechnetate..]..[.29. The methodof claim 19 wherein following step b) a second Sn (II) agent is added tothe combination of the completing agent and protein with Sn(II)-containing and sulfur-containing complexes in a sufficient mount tocompletely reduce the radionuclide, whereby the reduced radionuclideforms radionuclide-containing and sulfur-containing complexes..]..[.30.The method of claim 29 wherein the source of the second Sn (II) agent ispresent in a solution comprising alkali metal tartrate having a pH ofbetween approximately 5.0 and 6.0..]..[.31. The method of claim 29wherein the source of the second Sn (II) agent comprises a memberselected from the group consisting of stannous glucoheptonate, stannousgluconate and stannous phosphonate..]..[.32. The method of claim 29wherein following the addition of the second Sn (II) agent and prior toradiolabeling, the free Sn complexes, second Sn (II) agent and proteinwith Sn (II)-containing and sulfur-containing complexes are frozen in avial, whereby the frozen free Sn complexes, second Sn (II) agent andprotein with Sn (II)-containing and sulfur-containing complexes can bemaintained for an indefinite period before radiolabeling by the additionof the radionuclide to the vial..]..[.33. The method of claim 29 whereinfollowing the addition of the second Sn (II) agent and prior toradiolabeling, the free Sn complexes, second Sn (II) agent and proteinwith Sn (II)-containing and sulfur-containing complexes are lyophilizedin a vial, whereby the lyophilized free Sn complexes, second Sn (II)agent and protein with Sn (II)-containing and sulfur-containingcomplexes can be maintained for an indefinite period beforeradiolabeling by the addition of the radionuclide to the vial..]..[.34.The method of claim 29 wherein 85 percent or more of the radionuclide isstrongly bonded to the protein..].35. A method of radiolabeling aprotein containing monosulfide or disulfide bonds with a radionuclide toobtain stable labeling, comprising the steps of:a) incubating theprotein containing monosulfide or disulfide bonds with a first reducingagent, the period of incubation being sufficient to reduce availabledisulfide bonds to reactive sulfide groups while preventing excessivefragmentation of the protein; b) adding a source of first Sn (II) agentto the reduced protein to allow formation of Sn (II)-containing andsulfur-containing complexes; c) purifying the reduced protein with Sn(II)-containing and sulfur-containing complexes to substantially removethe first reducing agent and impurities; and d) radiolabeling thepurified reduced protein with Sn (II)-containing and sulfur-containingcomplexes by adding the radionuclide, whereby the complexed Sn(II) agentreduces the radionuclide and the reduced radionuclide and reducedprotein form radionuclide-containing and sulfur-containing complexes.36. The method of claim 35 wherein the radionuclide comprises a memberselected from the group consisting of technetium and rhenium.
 37. Themethod of claim 35, wherein the radionuclide is technetium-99m in theform of sodium pertechnetate.
 38. The method of claim
 35. wherein thefirst reducing agent comprises at least one member selected from thegroup consisting of 2-mercaptoethanol; 1,4-dithiothreitol,2,3-dihydroxybutane-1,4-dithiol, 2-aminoethanethiol HCl;2-mercaptoethylamine; thioglycolate; cyanide; and cysteine.
 39. Themethod of claim 35, wherein the source of the first Sn (II) agent ofstep b) is present in a solution comprising alkali metal tartrate havinga pH of between approximately 5.0 and 6.0.
 40. The method of claim 35wherein the protein comprises a member selected from the groupconsisting of monoclonal antibodies, monoclonal antibody fragments andpolyclonal antibodies.
 41. The method of claim 35 wherein following stepc), and prior to step d), the purified reduced protein with Sn(II)-containing and sulfur-containing complexes are frozen in a vial,whereby the purified reduced protein with Sn (II)-containing andsulfur-containing complexes can be maintained for an indefinite periodbefore radiolabeling in step d) by the addition of the radionuclide tothe vial.
 42. The method of claim 35 wherein following step c), andprior to step d), the purified reduced protein with Sn (II)-containingand sulfur-containing complexes are lyophilized in a vial, whereby thelyophilized purified reduced protein with Sn (II)-containing andsulfur-containing complexes can be maintained for an indefinite periodbefore radiolabeling in step d) by the addition of the radionuclide tothe vial.
 43. The method of claim 35 wherein 85 percent or more of theradionuclide is strongly bonded to the protein. The method of claim 35wherein following step c) a second reducing agent is added to thepurified reduced protein with Sn (II)-containing and sulfur-containingcomplexes in a sufficient mount to completely reduce the radionuclide,whereby the reduced radionuclide forms radionuclide-containing andsulfur-containing complexes.
 45. The method of claim 44 wherein thesecond reducing agent comprises a source of the Sn (II) agent present ina solution comprising alkali metal tartrate having a pH of betweenapproximately 5.0 and 6.0.
 46. The method of claim 44 wherein the secondreducing agent comprises a member selected from the group consisting ofstannous glucoheptonate, stannous gluconate and stannous phosphonate.47. The method of claim 44 wherein following the addition of the secondreducing agent and prior to radiolabeling, the second reducing agent andpurified reduced protein with Sn (II)-containing and sulfur-containingcomplexes are frozen in a vial, whereby the second reducing agent andpurified reduced protein with Sn (II)-containing and sulfur-containingcomplexes can be maintained for an indefinite period beforeradiolabeling by the addition of the radionuclide to the vial.
 48. Themethod of claim 44 wherein following the addition of the second reducingagent and prior to radiolabeling, the second reducing agent and purifiedreduced protein with Sn (II)-containing and sulfur-containing complexesare lyophilized in a vial, whereby the lyophilized second Sn reducingagent and purified reduced protein with Sn (II)-containing andsulfur-containing complexes can be maintained for an indefinite periodbefore radiolabeling by the addition of the radionuclide to the vial.49. The method of claim 44 wherein 85 percent or more of theradionuclide is strongly bonded to the protein. .Iadd.50. A method ofradiolabeling a whole antibody or F(ab')₂ antibody fragment containingone or more disulfide bonds with a radionuclide to obtain stablelabeling, comprising the steps of:a) incubating the antibody or fragmentwith a first Sn (II) agent, the period of incubation being sufficient toreduce a portion of the disulfide bonds and to allow formation of Sn(II)-containing and sulfur-containing complexes, while preventingexcessive fragmentation of the antibody or fragment; b) thereaftercomplexinh the free Sn agents with a subsequently addedpolyaminocarboxylic acid complexing agent so as not to further reducethe antibody or fragment, while retaining complexed Sn agents forreducing radionuclide, the radionuclide to be added in a subsequentstep; and c) radiolabeling the antibody or fragment with the Sn(II)-containing and sulfur-containing complexes by adding theradionuclide, whereby the complexed Sn (II) agents reduce theradionuclide and the reduced radionuclide forms radionuclide-containingand sulfur-containing complexes. .Iaddend..Iadd.51. The method of claim50 wherein the polyaminocarboxylic acid comprises at least one memberselected from the group consisting of EDTA and DTPA. .Iaddend..Iadd.52.The method of claim 50 wherein the source of the first Sn (II) agent ispresent in a solution comprising alkali metal tartrate having a pH ofbetween approximately 5.0 and 6.0. .Iaddend..Iadd.53. The method ofclaim 50 wherein the antibody or fragment is a member selected from thegroup consisting of monoclonal antibodies, monoclonal F(ab')₂ fragmentsand polyclonal antibodies. .Iaddend..Iadd.54. The method of claim 50wherein following step b), and prior to step c), the free Sn complexesand antibody or fragment with Sn(II)-containing and sulfur-containingcomplexes are frozen in a vial, whereby the frozen free Sn complexes andantibody or fragment with Sn (II)-containing and sulfur-containingcomplexes can be maintained for an indefinite period beforeradiolabeling in step d) by the addition of the radionuclide to thevial. .Iaddend..Iadd.55. The method of claim 50 wherein following stepb), and prior to step c), the free Sn complexes and antibody or fragmentwith Sn (II)-containing and sulfur-containing complexes are lyophilizedin a vial, whereby the lyophilized free Sn complexes and antibody orfragment with Sn (II)-containing and sulfur-containing complexes can bemaintained for an indefinite period before radiolabeling in step d) byaddition of the radionuclide to the vial. .Iaddend..Iadd.56. The methodof claim 50 wherein 85 percent or more of the radionuclide is stronglybonded to the antibody or fragment. .Iaddend..Iadd.57. The method ofclaim 50 wherein the radionuclide comprises a member selected from thegroup consisting of technetium and rhenium. .Iaddend..Iadd.58. Themethod of claim 50 wherein the radionuclide is technetium-99m in theform of sodium pertechnetate. .Iaddend..Iadd.59. The method of claim 50wherein following step b) a second Sn (II) agent is added to thecombination of the complexing agent and antibody or fragment with Sn(II)-containing and sulfur-containing complexes in a sufficient amountto completely reduce the radionuclide, whereby the reduced radionuclideforms radionuclide-containing and sulfur-containing complexes..Iaddend..Iadd.60. The method of claim 59 wherein the source of thesecond Sn (II) agent is present in a solution comprising alkali metaltartrate having a pH of between approximately 5.0 and 6.0..Iaddend..Iadd.61. The method of claim 59 wherein the source of thesecond Sn (II) agent comprises a member selected from the groupconsisting of stannous glucoheptonate, stannous gluconate and stannousphosphonate. .Iaddend..Iadd.62. The method of claim 59 wherein followingthe addition of the second Sn (II) agent and prior to radiolabeling, thefree Sn complexes, second Sn (II) agent and antibody or fragment with Sn(II)-containing and sulfur-containing complexes are frozen in a vial,whereby the frozen free Sn complexes, second Sn (II) agent and antibodyor fragment with Sn (II)-containing and sulfur-containing complexes canbe maintained for an indefinite period before radiolabeling by theaddition of the radionuclide to the vial. .Iaddend..Iadd.63. The methodof claim 59 wherein following the addition of the second Sn (II) agentand prior to radiolabeling, the free Sn complexes, second Sn (II) agentand antibody or fragment with Sn (II)-containing and sulfur-containingcomplexes are lyophilized in a vial whereby the lyophilized free Sncomplexes, second Sn (II) agent and antibody or fragment with Sn(II)-containing and sulfur-containing complexes can be maintained for anindefinite period before radiolabeling by the addition of theradionuclide to the vial. .Iaddend..Iadd.64. The method of claim 59wherein 85 percent or more the radionuclide is strongly bonded to theantibody or fragment. .Iaddend.